The RAS signaling pathway controls cell growth, survival and death in all multicellular organisms. Deregulation of RAS proto-oncogenes is found in approximately 30% of human cancer. Thus an important goal in cancer biology is to understand the molecular mechanisms underlying normal and aberrant RAS signals. Drosophila R7 photoreceptor cell fate specification is a genetically tractable model in which to study RAS signal transduction. In this system, we have shown that RAS activation directs a ubiquitin-mediated proteolysis pathway mediated by SEVEN-IN-ABSENTIA (SINA). SINA encodes a member of a highly conserved family of E3 ubiquitin ligases and has been implicated in neuronal differentiation, apoptosis, stress response, tumor suppression, beta-catenin, APC and p53 signaling in vertebrates. The substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligases, and the focus of our proposal is the SINA E3 ligase, an essential downstream component of RAS signaling pathway in Drosophila. The objectives of this research proposal are (1) to determine the developmental roles and regulation of SINA E3 family-dependent proteolysis in RAS signaling using Drosophila as a model organism, (2) to identify new SINA pathway components and determine their function in regulated proteolysis in RAS signal transduction, and (3) to investigate the roles of the mammalian SINA homologues (SIAHs) in RAS-mediated oncogenesis and to elucidate how the SIAH-dependent proteolysis promotes cell growth, differentiation and apoptosis during animal development and human cancers. Our central hypothesis is that the SINA-dependent proteolysis plays an important role in development. We will test this hypothesis by examining sina loss-of function phenotypes, by identifying new SINA pathway components by genetic screens and biochemical purification, and by determining their functions by a combination of genetic, molecular and biochemical analyses. We will also test the hypothesis that SIHA is required for RAS signal transduction during oncogenesis. We have detected enhanced expression of SINA in rapidly dividing cells during normal mammalian development and in human carcinomas, suggesting that SINA may be required for RAS-mediated cell proliferation in carcinogenesis. We propose to identify the function and regulation of SINA proteins in Drosophila, and apply what we have learned to human pancreatic cancer cells. Since many known SINA substrates have a demonstrated role in oncogenesis, it is likely that the proposed research will identify novels targets for anticancer therapies and contribute to our understanding of cancer biology.